C/EBPBeta and Elk-1 synergistically transactivate the c-fos serum response element

BACKGROUND: The serum response element (SRE) in the c-fos promoter is a convergence point for several signaling pathways that regulate induction of the c-fos gene. Many transcription factors regulate the SRE, including serum response factor (SRF), ternary complex factor (TCF), and CCAAT/enhancer binding protein-beta (C/EBPβ). Independently, the TCFs and C/EBPβ have been shown to interact with SRF and to respond to Ras-dependent signaling pathways that result in transactivation of the SRE. Due to these common observations, we addressed the possibility that C/EBPβ and Elk-1 could both be necessary for Ras-stimulated transactivation of the SRE. RESULTS: In this report, we demonstrate that Elk-1 and C/EBPβ functionally synergize in transactivation of both a Gal4 reporter plasmid in concert with Gal4-SRF and in transactivation of the SRE. Interestingly, this synergy is only observed upon activation of Ras-dependent signaling pathways. Furthermore, we show that Elk-1 and C/EBPβ could interact both in an in vitro GST-pulldown assay and in an in vivo co-immunoprecipitation assay. The in vivo interaction between the two proteins is dependent on the presence of activated Ras. We have also shown that the C-terminal domain of C/EBPβ and the N-terminal domain of Elk-1 are necessary for the proteins to interact. CONCLUSIONS: These data show that C/EBPβ and Elk-1 synergize in SRF dependent transcription of both a Gal-4 reporter and the SRE. This suggests that SRF, TCF, and C/EBPβ are all necessary for maximal induction of the c-fos SRE in response to mitogenic signaling by Ras

Molecular study of SRF-cofactor interactions.

Serum Response Factor regulates a large array of genes involved in diverse processes including cell proliferation, muscle differentiation and development, and cytoskeletal processes such as cell migration and adhesion. The specificity and versatility of the SRF responses is achieved by combinatorial interactions with accessory factors. SRF binds to the CC(A/T)2A(A/T)3GG CArG box consensus sequence within the promoters of its target genes and acts as a docking platform for diverse signal regulated and cell- type specific cofactors to elicit their distinct responses. In fibroblasts two pathways signal through SRF in a mutually exclusive manner. MAP kinase signalling results in transcriptional activation of a subset of SRF target genes, via the interaction of SRF with members of the TCF family of Ets domain proteins. In contrast Rho-signalling induced changes in actin dynamics result in the association of SRF with members of the Myocardin-related family of SRF cofactors (MAL/MRTF-A/MKL1 and MAL16/MRTF-B/MKL2). The results described in this thesis characterise the molecular mechanism of MAL-SRF complex formation. MAL binds SRF as a dimer via a seven-residue core sequence within the MAL B1 region. Residues in the neighbouring Q-box enhance MAL-SRF complex formation, although these do not contact SRF directly. The MAL-SRF interaction displays the properties of a Rho-regulated cofactor. MAL competes with TCF for SRF binding due to the interaction of both cofactors with the same hydrophobic groove and pocket on SRF. In contrast to TCF, MAL-SRF complex formation depends on the intact N-terminus of the SRF DNA-binding domain. Mutations in the SRF al-helix that reduce DNA bending also impair complex formation with MAL. These mutations however do not affect DNA distortion in the MAL-SRF complex. Efficient MAL-SRF complex formation requires that SRF be bound to its cognate DNA and that MAL directly contacts DNA on either side of the CArG box. My results support a model in which each MAL monomer adds a p-strand consisting of the core B1 sequence, to the p-sheet of the SRF DNA-binding domain in a similar way to TCF, while also making direct DNA contacts in the ternary complex facilitated by SRF- induced DNA distortion. My analysis of complex formation between MAL and SRF demonstrates that members of the MRTF and TCF families of SRF cofactors interact with SRF using related but distinct mechanisms, thus providing a molecular rationale for their mutually exclusive transcriptional responses and the specificity of signalling to SRF

Serum Regulation of Inhibitor of DNA Binding/Differentiation 1 Expression by a BMP Pathway and BMP Responsive El

Immediate Early Genes (IEGs) are expressed upon re-entry of quiescent cells into the cell cycle following serum stimulation. These genes are involved in growth control and differentiation and hence their expression is tightly controlled. Many IEGs are regulated through Serum Response Elements (SREs) in their promoters, which bind Serum Response Factor (SRF). However, many other IEGs do not have SREs in their promoters and their serum regulation is poorly understood. We have identified SRF-independent IEGs in SRF-depleted fibroblasts. One of these, Id1, was examined more closely. We mapped a serum responsive element in the Id1 promoter and find that it is identical to a BMP Responsive Element (BRE). The Id1 BRE is necessary and sufficient for the serum regulation of Id1. Inhibition of the BMP pathway by siRNA depletion of Smad4, treatment with the BMP antagonist noggin, or the BMP receptor inhibitor dorsomorphin blocked serum induction of Id1. Further, BMP2 is sufficient to induce Id1 expression. Given reports that SRC inhibitors can block Id1 expression, we tested the SRC inhibitor, AZD0530, and found that it inhibits the serum activation of Id1. Surprisingly, this inhibition is independent of SRC or its family members. Rather, we show that AZD0530 directly inhibits the BMP type I receptors. Serum induction of the Id1 related gene Id3 also required the BMP pathway. Given these and other findings we conclude that the Id family of IEGs is regulated by BMPs in serum through similar BREs. This represents a second pathway for serum regulation of IEGs

Molecular dynamics simulations and in silico peptide ligand screening of the Elk-1 ETS domain

Background: The Elk-1 transcription factor is a member of a group of proteins called ternary complex factors, which serve as a paradigm for gene regulation in response to extracellular signals. Its deregulation has been linked to multiple human diseases including the development of tumours. The work herein aims to inform the design of potential peptidomimetic compounds that can inhibit the formation of the Elk-1 dimer, which is key to Elk-1 stability. We have conducted molecular dynamics simulations of the Elk-1 ETS domain followed by virtual screening. Results: We show the ETS dimerisation site undergoes conformational reorganisation at the a1b1 loop. Through exhaustive screening of di- and tri-peptide libraries against a collection of ETS domain conformations representing the dynamics of the loop, we identified a series of potential binders for the Elk-1 dimer interface. The di-peptides showed no particular preference toward the binding site; however, the tri-peptides made specific interactions with residues: Glu17, Gln18 and Arg49 that are pivotal to the dimer interface. Conclusions: We have shown molecular dynamics simulations can be combined with virtual peptide screening to obtain an exhaustive docking protocol that incorporates dynamic fluctuations in a receptor. Based on our findings, we suggest experimental binding studies to be performed on the 12 SILE ranked tri-peptides as possible compounds for the design of inhibitors of Elk-1 dimerisation. It would also be reasonable to consider the score ranked tri-peptides as a comparative test to establish whether peptide size is a determinant factor of binding to the ETS domain

ELK1 Uses Different DNA Binding Modes to Regulate Functionally Distinct Classes of Target Genes

Eukaryotic transcription factors are grouped into families and, due to their similar DNA binding domains, often have the potential to bind to the same genomic regions. This can lead to redundancy at the level of DNA binding, and mechanisms are required to generate specific functional outcomes that enable distinct gene expression programmes to be controlled by a particular transcription factor. Here we used ChIP–seq to uncover two distinct binding modes for the ETS transcription factor ELK1. In one mode, other ETS transcription factors can bind regulatory regions in a redundant fashion; in the second, ELK1 binds in a unique fashion to another set of genomic targets. Each binding mode is associated with different binding site features and also distinct regulatory outcomes. Furthermore, the type of binding mode also determines the control of functionally distinct subclasses of genes and hence the phenotypic response elicited. This is demonstrated for the unique binding mode where a novel role for ELK1 in controlling cell migration is revealed. We have therefore uncovered an unexpected link between the type of binding mode employed by a transcription factor, the subsequent gene regulatory mechanisms used, and the functional categories of target genes controlled

Characterization of self-regulatory mechanisms and internal dynamics of ETS transcription factor PU.1

The ETS family of transcription factors bind to site-specific DNA via DNA-binding domains called the ETS domains. The ETS domains are structurally homologous but divergent in primary sequences. PU.1 is an essential transcription factor and its biological activity is primarily controlled by up- and down-regulation of its expression. Aside from down-regulated expression, only a few inhibitory mechanisms are known for PU.1. The most understood one involves PU.1 forming a heterodimer with other protein partners, such as GATA-1. However, unlike auto-inhibited ETS-family members whose activity is regulated by autoinhibitory elements that reduce the net affinity of binding to specific DNA, PU.1 has no such regulatory mechanism at the protein-DNA level. We report here that PU.1, unlike its auto-inhibited paralog Ets-1, forms a 2:1 complex with site-specific DNA (\u3e10 bp) in a negatively cooperative manner. We also detected potential interface (193DKDK196) of the PU.1 dimer by using heteronuclear single quantum correlation (HSQC) NMR. Self-titration of PU.1 is a negative feedback mechanism at the protein-DNA level. Following these findings, our group found the presence of the IDRs flanking the ETS domain does not change the DNA binding modes of the PU.1 ETS domain, yet the PEST domain modifies DNA recognition by the ETS domain through changing DNA binding affinities. We successfully assigned ~90% or more backbone amide resonances in the 1H-15N HSQC spectra of hPU.1 constructs with and without IDRs, in the absence and presence (1:1 complex) of DNA. Using the fully assigned HSQC spectra, we studied fast (ps to ns) time scale internal dynamics of PU.1 protein. Spin relaxation rates and heteronuclear 1H{15N}-NOE were acquired for the hPU.1 proteins with and without DNA by NMR. We demonstrated that the PEST domain remains disordered but becomes more dynamic upon specific DNA binding. In terms of DNA recognition, the presence of the PEST domain increases the affinity of 1:1 complex of the ETS domain with cognate DNA, without perturbing the structure or changing the fast time scale backbone motions of the ETS domain

Mechanisms of transcriptional regulation by SRF co-factors

Serum response factor (SRF) controls gene activation in response to changes in actin dynamics and mitogen-activated protein kinases. SRF has low intrinsic transcriptional activity and requires the recruitment of one of two families of co-activators: the MRTFs (myocardin-related transcription factors) and the TCFs (ternary complex factors). MRTFs are actin-binding proteins. Disruption of the actin-MRTF interaction is sufficient to induce MRTF nuclear accumulation and transcriptional activation. The TCF family are specifically activated by MAPK signalling. This thesis aims to elucidate how the SRF transcription network is controlled. The work presented encompasses two projects focused on each of the co-activator families. The regulation of MRTF shuttling from the cytoplasm to the nucleus is relatively well understood while its regulation once in the nucleus is still uncharacterized. The work demonstrates that nuclear MRTF activities are influenced by nuclear actin. Nuclear actin interferes with MRTF-DNA binding and target gene activation. In the presence of G-actin, nuclear MRTF can associate with target loci and recruit Pol II that, although traverses the gene body, does not generate stable mRNA. This inhibited state is accompanied by hypo-phosphorylation of the Pol II CTD. Dissociation of MRTF-actin interaction is required to re-establish Pol II phosphorylation and mRNA accumulation. The Erk-TCF signalling pathway was used to investigate how chromatin signatures are established in response to cues. Fibroblasts lacking all three TCFs, or reconstituted with mutant derivatives of the Elk-1 TCF were generated. Following Erk activation, chromatin immunoprecipitation and RNA-sequencing techniques, were employed to study the role of the TCFs in chromatin changes and transcriptional activation. It was possible to show that signal-induced chromatin changes occur in absence of transcription, and the specific chromatin signature requires Elk-1 DNA binding and phosphorylation. In addition analysis of the H3K27me3 mark demonstrated that Elk-1 activation is required to maintain a permissive chromatin landscape

Comparative analysis of protein expression systems and PTM landscape in the study of transcription factor ELK-1

Post-translational modifications (PTMs) are important for protein folding and activity, and the ability to recreate physiologically relevant PTM profiles on recombinantly-expressed proteins is vital for meaningful functional analysis. The ETS transcription factor ELK-1 serves as a paradigm for cellular responses to mitogens and can synergise with androgen receptor to promote prostate cancer progression, although in vitro protein function analyses to date have largely overlooked its complex PTM landscapes. We expressed and purified human ELK-1 using mammalian (HEK293T), insect (Sf9) and bacterial (E. coli) systems in parallel and compared PTMs imparted upon purified proteins, along with their performance in DNA and protein interaction assays. Phosphorylation of ELK-1 within its transactivation domain, known to promote DNA binding, was most apparent in protein isolated from human cells and accordingly conferred the strongest DNA binding in vitro, while protein expressed in insect cells bound most efficiently to the androgen receptor. We observed lysine acetylation, a hitherto unreported PTM of ELK-1, which appeared highest in insect cell-derived ELK-1 but was also present in HEK293T-derived ELK-1. Acetylation of ELK-1 was enhanced in HEK293T cells following starvation and mitogen stimulation, and modified lysines showed overlap with previously identified regulatory SUMOylation and ubiquitination sites. Our data demonstrate that the choice of recombinant expression system can be tailored to suit biochemical application rather than to maximise soluble protein production and suggest the potential for crosstalk and antagonism between different PTMs of ELK-1

The role of monoubiquitylation in the regulation of the transcription factor Elk-1

Eukaryotic cells respond to extracellular stimuli by transmitting intracellular instructions via signalling pathways to coordinate appropriate responses. Mitogen-activated protein kinase (MAPK) pathways are often used to transmit these instructions to regulate gene expression, where Ternary Complex Factors (TCFs) are among their nuclear targets. Elk-1 is the founding member of the TCF family of transcription activators. The mechanism of function and regulation of Elk-1 has been extensively studied, therefore providing a paradigm for signal-induced transcription. The activity of Elk-1 is influenced by Post-Translational Modifications (PTMs), such as phosphorylation and sumoylation. Elk-1 ubiquitylation has also been reported in vitro, however little work has been done on this modification of Elk-1. This thesis sought to reveal the mechanism of regulation and function of Elk-1 ubiquitylation. Elk-1 was demonstrated to be both monoubiquitylated and polyubiquitylated in vitro and in cells. Using size exclusion chromatography and the dominant negative nedd8 conjugating E2 enzyme Ubc12, several features of the Elk-1 specific E3 ligases have been revealed in vitro. In cells, ternary complex formation was shown to be important for monoubiquitylation. Furthermore monoubiquitylated Elk-1 is diminished following ERK-mediated phosphorylation, hence activation, in response to mitogen stimulation. It was also demonstrated that an Elk-1 derivative that exhibits strong monoubiquitylation level also exhibits a reduced capability to transactivate gene expression at the Serum Responsive Element (SRE), indicating a negative role of monoubiquitylation on Elk-1 transcriptional ability

De-ubiquitination of ELK-1 by USP17 potentiates mitogenic gene expression and cell proliferation

ELK-1 is a transcription factor involved in ERK-induced cellular proliferation. Here we show that its transcriptional activity is modulated by ubiquitination at lysine 35 (K35). The level of ubiquitinated ELK-1 rises in mitogen-deprived cells and falls upon mitogen stimulation or oncogene expression. Ectopic expression of USP17, a cell cycle-dependent deubiquitinase, decreases ELK-1 ubiquitination and up-regulates ELK-1 target-genes with a concomitant increase in cyclin D1 expression. In contrast, USP17 depletion attenuates ELK-1-dependent gene expression and slows cell proliferation. The reduced rate of proliferation upon USP17 depletion appears to be a direct effect of ELK-1 ubiquitination because it is rescued by an ELK-1(K35R) mutant refractory to ubiquitination. Overall, our results show that ubiquitination of ELK-1 at K35, and its reversal by USP17, are important mechanisms in the regulation of nuclear ERK signalling and cellular proliferation. Our findings will be relevant for tumours that exhibit elevated USP17 expression and suggest a new target for intervention